Most of what we know about antifungal drug research is derived from studies of nonpathogenic model fungi, such as Saccharomyces cerevisiae, which can easily be inserted into high throughput screening platforms, but are unsuitable as pan-fungal surrogates. Alternatively, species-specific antifungal development is unrealistic due to cost and lack of market return on research investment. The rapid completion of genome sequences, and continually evolving bioinformatic manipulation of this vast and growing amount of data, has enabled new approaches to antifungal development that can now start with drug targets, and work backwards towards drug development-a method referred to as rational design. This strategy has one important requirement; the need for biologically essential targets to design drugs against so that the drug is lethal to the pathogen. Because of this requirement, the major objective of this study will be to develop a way to rapidly and inexpensively identify these targets. To accomplish this goal, we will utilize two taxonomically diverged yeasts, Cryptococcus neoformans (a basidiomycete) and Candida albicans (an ascomycete) that have working Agrobacterium tumefaciens (AT) transformation systems. The first aim will be to improve the existing AT transformation efficiency to yield enough transformants to produce a saturated insertional mutagenesis map. We will next develop a capture-probe based enrichment method for recovering insertion site fragments from the predominating non-junctional genomic DNA background. Finally, we will apply deep sequencing to these enriched fragments to identify each insertion site and its neighboring flanking genomic DNA, and ultimately assemble a high density insertion map that will be used to identify which genes are essential. The future long-term goal that this proposal will enable with subsequent studies will be the identification of a core set of essential genes that all fungi have. This core set of genes can then serve as a target group for broad spectrum antifungal development.